Canine Mucosal Artificial Colon: development of a new colonic in vitro model adapted to dog sizes

Abstract

Differences in dog breed sizes are an important determinant of variations in digestive physiology, mainly related to the large intestine. In vitro gut models are increasingly used as alternatives to animal experiments for technical, cost, societal, and regulatory reasons. Up to now, only one in vitro model of the canine colon incorporates the dynamics of different canine gut regions, yet no adaptations exist to reproduce size-related digestive parameters. To address this limitation, we developed a new model of the canine colon, the CANIne Mucosal ARtificial COLon (CANIM-ARCOL), simulating main physiochemical (pH, transit time, anaerobiosis), nutritional (ileal effluent composition), and microbial (lumen and mucus-associated microbiota) parameters of this ecosystem and adapted to three dog sizes (i.e., small under 10 kg, medium 10–30 kg, and large over 30 kg). To validate the new model regarding microbiota composition and activities, in vitro fermentations were performed in bioreactors inoculated with stools from 13 dogs (4 small, 5 medium, and 4 large). After a stabilization period, microbiota profiles clearly clustered depending on dog size. Bacteroidota and Firmicutes abundances were positively correlated with dog size both in vitro and in vivo, while opposite trends were observed for Actinobacteria and Proteobacteria. As observed in vivo, microbial activity also increased with dog size in vitro, as evidenced from gas production, short-chain fatty acids, ammonia, and bile acid dehydroxylation. In line with the 3R regulation, CANIM-ARCOL could be a relevant platform to assess bilateral interactions between food and pharma compounds and gut microbiota, capturing inter-individual or breed variabilities. Key points • CANIM-ARCOL integrates main canine physicochemical and microbial colonic parameters • Gut microbiota associated to different dog sizes is accurately maintained in vitro • The model can help to move toward personalized approach considering dog body weight

Large intestinal nutritional and physicochemical parameters from different dog sizes reshape canine microbiota structure and functions in vitro

“…In a previous study, we developed a new in vitro gut model, the CANIM-ARCOL, reproducing the main parameters of the canine colonic ecosystem and adapted to three dog sizes, i.e. small, medium, and large sizes [ 26 ]. The model was set up to reproduce the main physicochemical, nutritional, and microbial parameters specific to each dog size and inoculated with fecal samples from size-related conditions (e.g.…”

“…With this objective in mind, we first performed comparisons on the effect of dog size between the present results and those previously obtained in the CANIM-ARCOL inoculated with fecal samples from small, medium, and large dogs [ 26 ], as described in Table 4 . We should keep in mind that, even if there are of interest, such comparisons should be hampered by differences between the two studies in time of fermentation (9 days here versus 21 days in the previous study) and number of fecal samples (2 dogs versus 13 dogs).…”

“…CANIM-ARCOL is a one-stage fermentation system (MiniBio, Applikon, Delft, The Netherlands), inoculated with stool samples and used under continuous conditions to simulate the nutritional, physicochemical, and microbial conditions found in the large intestine of dogs, as previously described in [ 26 ]. Briefly, the in vitro model is composed of a main bioreactor simulating the colonic luminal medium and an airtight glass vessel connected to this bioreactor and containing mucin beads to reproduce the mucosal compartment ( Figure 1a ).…”

“…Every 2 days, mucin beads from the mucosal compartment were renewed by fresh sterile ones under a constant flow of CO 2 to avoid oxygen entrance, as previously described [ 27 ]. In the present study, the CANIM-ARCOL was set up with nutritional and physicochemical parameters adapted to three dog sizes as previously validated and detailed in Table 2 [ 26 ].

Figure 1.

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“…This alternative strategy is fully in line with European ‘3 R’ rules and provides cost, technical, ethical, and regulatory benefits compared to in vivo assays [ 23 , 24 ]. Two dynamic models of the canine colon have been recently developed, namely the Mucosal Simulator of the Canine Intestinal Microbial Environment (M-SCIME) [ 25 ] and the Canine Mucosal Artificial Colon (CANIM-ARCOL) [ 26 ]. Only the second one has been adapted to simulate the specific conditions (i.e.…”